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The cell membrane is a complex and highly regulated system that is composed of lipid bilayer and proteins. One of the main functions of the cell membrane is the regulation of cell entry. Cell-penetrating peptides (CPPs) are defined as peptides that can cross the plasma membrane and deliver their cargo inside the cell. The uptake of a peptide is determined by its sequence and biophysicochemical properties. At the same time, the uptake mechanism and efficiency are shown to be dependent on local peptide concentration, cell membrane lipid composition, characteristics of the cargo, and experimental methodology, suggesting that a highly efficient CPP in one system might not be as productive in another. To better understand the dependence of CPPs on the experimental system, we present a review of the in vitro assays that have been employed in the literature to evaluate CPPs and CPP-cargos. Our comprehensive review suggests that utilization of orthogonal assays will be more effective for deciphering the true ability of CPPs to translocate through the membrane and enter the cell cytoplasm.

In Vitro Assays: Friends or Foes of Cell-Penetrating Peptides